Prion Cycle Regulation In Vivo

描述（由申请人提供）：朊病毒假说为一系列先前无法解释的现象提供了解释，这些现象包括哺乳动物神经退行性疾病的出现、进展和传播，以及真菌独特性状的非孟德尔遗传。根据这一观点，当一种蛋白质（称为朊病毒）采用另一种物理状态并在自我复制时持续存在时，朊病毒相关表型就会出现。这种自我复制是由交替折叠的朊病毒组装成聚集体介导的，聚集体作为模板将其他形式的蛋白质转化为类似状态。朊病毒利用其构象灵活性的能力是建立不同表型的核心事件，但这一过程在活细胞中是一个多步骤的奋进。蛋白质质量控制途径、朊病毒生物发生和细胞生物学改变朊病毒在体内的折叠以产生生理学上的可传递变化。这些力量如何相交的分子理解是目前知识的差距，限制了我们的能力，相关的朊病毒折叠机制在体外和它们的生理后果在体内。由于朊病毒相关表型之间的转换必然涉及蛋白质状态的变化，这些力量必须集中在体内调节朊病毒动力学的事件上。本研究的长期目标是阐明影响朊病毒折叠的细胞机制，以产生生理学上的可传播变化。本申请的总体目标是确定朊病毒折叠途径、细胞质量控制和细胞生物学的其它方面结合联合收割机以产生朊病毒相关表型的分子机制。我们的方法是确定Sup 35序列，构象和表达水平的变化改变朊病毒在体内繁殖的途径。中心假设是Sup 35的物理特性和丰度调节了分子伴侣识别和/或处理朊病毒形式的效率，从而允许不同的表型出现并持续存在，但偶尔也会相互转化。在使用实验追踪的S.为了验证这一假设，将通过三个特定的目的来检验：1）确定Sup 35的序列变体改变[PSI+]繁殖的分子机制，2）确定过量的Hsp 104导致[PSI+]损失的分子机制，和3）确定Sup 35聚集产生[PSI+]表型的分子机制。这些拟议的研究是创新的，因为它们使用了实验和数学分析的独特组合来测试朊病毒相关表型的新的动态模型。拟议的研究是重要的，因为它解决了朊病毒折叠的细胞调节，这是一个知之甚少但重要的因素，允许朊病毒假说在酵母中创建基于蛋白质的表观遗传元件。在实验上易于处理的酵母系统中获得的这一知识有可能提供新的假设，这些假设可以在涉及朊病毒机制的更复杂的系统中进行测试。 公共卫生相关性：由于朊病毒蛋白质的错误折叠与人类的家族性、散发性和传染性神经变性疾病有关，因此拟议的研究与公共卫生有关。因此，在本发明中，我们提出的研究与NIH的使命相关，因为在此获得的知识有可能为理解疾病提供新的框架人的动力学

DESCRIPTION (provided by applicant): The prion hypothesis provides an explanation for a collection of previously inexplicable phenomena, ranging from the appearance, progression and spread of mammalian neurodegenerative disease to the non-Mendelian inheritance of unique traits in fungi. According to this idea, prion-associated phenotypes arise when a protein, known as a prion, adopts an alternative physical state and persist when that form self-replicates. This self- replication is mediated by the assembly of alternatively folded prions into aggregates, which template the con- version of other forms of the protein to a like state. The ability of prions to harness their conformational flexibil- ity is a central event in establishing distinct phenotypes, but this process becomes a multistep endeavor within the context of a living cell. Protein quality control pathways, prion biogenesis, and cell biology modify prion folding in vivo to create transmissible changes in physiology. A molecular understanding of how these forces intersect is a gap in current knowledge, limiting our ability to correlate prion folding mechanisms in vitro and their physiological consequences in vivo. As transitions between prion-associated phenotypes necessarily in- volve changes in protein state, these forces must converge on events that regulate prion dynamics in vivo. The long-term goal of this research is to elucidate the cellular mechanisms that influence prion folding to pro- duce transmissible changes in physiology. The overall objective of this application is to determine the molecu- lar mechanisms through which the prion folding pathway, cellular quality control, and other aspects of cell biol- ogy combine to create prion-associated phenotypes. Our approach is to determine the pathways through which variations in Sup35 sequence, conformation, and expression levels alter prion propagation in vivo. The central hypothesis is that the physical characteristics and abundance of Sup35 temper the efficiency with which mo- lecular chaperones recognize and/or process the prion form, thereby allowing distinct phenotypes to arise and persist but also to occasionally interconvert. Guided by strong preliminary data using the experimentally trac- table Sup35/[PSI+] prion of S. cerevisiae, this hypothesis will be tested through three specific aims: 1) Deter- mine the molecular mechanism by which sequence variants of Sup35 alter [PSI+] propagation, 2) Determine the molecular mechanism by which excess Hsp104 leads to [PSI+] loss, and 3) Determine the molecular mechanism by which Sup35 aggregation creates the [PSI+] phenotype. These proposed studies are innovative because they use a unique combination of experimental and mathematical analyses to test a new and dynamic model for prion-associated phenotypes. The proposed research is significant because it addresses the cellular regulation of prion folding, a poorly understood but significant factor that allows the prion hypothesis to create protein-based epigenetic elements in yeast. This knowledge, gained in the experimentally tractable yeast sys- tem, has the potential to provide new hypotheses that can be tested in more complex systems where a prion mechanism has been implicated. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public heath because the misfolding of prion proteins is associated with a wide array of familial, sporadic and transmissible neurodegenerative disease in man. Our current understanding of how protein misfolding correlates with disease characteristics is limited; thus, our proposed studies are relevant to NIH's mission because the knowledge gained here has the potential to provide a new framework for understanding disease dynamics in man.